Rotavirus enterotoxin NSP4 and methods of using same

ABSTRACT

Methods of immunization against rotavirus infection or rotavirus disease by administering to a subject a peptide NSP4 114-135, a peptide NSP4 120-147, or a toxoid thereof are disclosed.

This application is the National Stage of International Application No.PCT/US96/10523, filed on Jun. 14, 1996, which claims the benefit of U.S.Provisional Application No. 60/000,220, filed Jun. 14, 1995 and a CIP ofU.S. Application No. 08/628,014, filed Apr. 4, 1996, now abandoned

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the viral enterotoxin NSP4 and to methods forusing it, or antibodies/antisera thereto, as diagnostic agents, vaccinesand therapeutic agents for the detection, prevention and/or treatment ofrotaviral disease, for the prevention of stunted growth in animals andchildren caused by rotaviral infection and for the treatment of cysticfibrosis. This invention also relates to methods and animal modelsfor 1) the screening for viral enterotoxins, 2) the detection of viralenterotoxins and 3) the identification of viral enterotoxins.

2. Related Technology

Rotaviruses are the leading cause of severe, life-threatening viralgastroenteritis in infants and animals (1) and are associated withsporadic outbreaks of diarrhea in elderly (2) and immunocompromisedpatients (3). These viruses have a limited tissue tropism, withinfection primarily being restricted to cells of the small intestine(4). Rotavirus infections also cause morbidity and mortality in manyanimal species. Moreover, the outcome of infection is age-related;although rotaviruses may infect individuals and animals of all ages,symptomatic infection (i.e., diarrhea) generally occurs in the young (6months-2 years in children, and up to 14 days in mice), and the elderly.

Age-related host factors which may influence the outcome of infectionhave been proposed to include 1) differences in the presence/quantity ofvirus-binding receptors on mature villus epithelial cells, 2) virusstrains with a specific spike protein (VP4), 3) passive immunityacquired by maternal antibody or in colostrum, and 4) reduced levels ofproteases in the young.

Disease resulting from rotavirus infection in mice has been studied moreextensively than in any other species and an age restriction of diseasehas been reported by several investigators (5). Only mice less than 14days of age develop diarrhea following oral inoculation of murinerotavirus, and the peak age at which animals are most likely to developdiarrhea (6-11 days) corresponds to the age when rotavirus can bind tomouse enterocytes (6). Treatment of 8 day old mice with cortisoneacetate which promotes premature maturation of intestinal epithelialcells, results in a reduced susceptibility to rotavirus-induceddiarrhea, although the mice can still be infected (7). These data wereinterpreted to suggest that the capacity of murine rotaviruses to inducediarrhea in young, but not adult mice, is due to the quantity ofrotavirus-binding receptors on the surface of villus epithelial cells inthe young mouse intestine.

When compared to rotavirus infections in other species, rotavirusinfections in mice show minimal histologic alterations. That is, villusblunting is limited and transient, and crypt cell hyperplasia is notpresent. In addition, the loss of villus tip epithelial cells is morelimited in mice than in other animals. Instead, vacuolization ofenterocytes on the villus tips is a predominant feature in symptomaticrotavirus infection in mice and virus replication may be abortive (8).The lack of extensive pathologic alterations in the mouse intestineduring symptomatic infections has remained a puzzle; one interpretationof this phenomenon is that a previously unrecognized mechanism ofdiarrhea induction may be active in symptomatic rotavirus infection inmice.

Despite the prevalence of rotavirus infections and extensive studies inseveral animal models and many advances in understanding rotavirusimmunity, epidemiology, replication and expression, rotaviruspathogenesis, specifically, the mechanism of diarrhea induction, remainspoorly understood. Proposed pathophysiologic mechanisms by whichrotaviruses induce diarrhea following viral replication and viralstructural protein synthesis include malabsorption secondary to thedestruction of enterocytes (9), disruption of transepithelial ionhomeostasis resulting in fluid secretion (10), and local villus ischemialeading to vascular damage and diarrhea (11). However, these proposedmechanisms do not explain cases of rotavirus-induced diarrhea observedprior to, or in the absence of, histopathologic changes (12).

On the other hand, the pathophysiology of bacterial-induced diarrheabased on interactions with intestinal receptors and bacterialenterotoxins is well understood (13). The heat-stable toxin A and theheat-labile toxin of E. coli, and guanylin (an endogenous, 15 amino acidintestinal ligand originally isolated from rat jejunum) induce diarrheaby binding a specific intestinal receptor, increasing cAMP or cGMP, andactivating a cyclic nucleotide signal transduction pathway (14). The neteffect of these bacterial toxins is to increase Cl⁻ secretion, anddecrease Na⁺ and water absorption.

Previous studies in insect cells indicated that a receptor-mediatedphospholipase C pathway is associated with the increases in [Ca²⁺]_(i),following exogenous treatment of cells with NSP4 or NSP4 114-135 peptide(15). The rotavirus nonstructural ER glycoprotein, NSP4, has been shownto have multiple functions including the release of calcium from theendoplasmic reticulum (ER) in SF9 insect cells infected with recombinantbaculovirus containing the NSP4 cDNA (15, 16). In addition, NSP4disrupts ER membranes and may play an important role in the removal ofthe transient envelope from budding particles during viral morphogenesis(unpublished data). NSP4 114-135, a 22 aa peptide of NSP4 protein, hasbeen shown to be capable of mimicking properties associated with NSP4including being able to (i) mobilize intracellular calcium levels ininsect cells when expressed endogenously or added to cells exogenously(15, 16), and (ii) destabilize liposomes. (unpublished data).

Expression of NSP4 in insect cells increased [Ca²⁺]_(i) levels from asubset of the thapsigargin-sensitive store (ER) (15). The [Ca²⁺]_(i)mobilized by NSP4 or the NSP4 114-135 peptide was blocked by aphospholipase C inhibitor, the U-73122 compound, suggesting that areceptor-mediated pathway is responsible for the calcium release fromthe ER induced by NSP4 (15).

BRIEF DESCRIPTION OF THE INVENTION

This invention stems from the discovery of the first known viralenterotoxin, rotavirus NSP4, previously called NS28, which encodes aviral toxin capable of inducing intestinal secretion through aheretofore unknown signal transduction pathway to cause diarrhealdisease.

This paper reports the fortuitous discovery that the rotavirusnonstructural ER glycoprotein, NSP4, induces an age-dependent diarrheain two rodent models. Induction of diarrhea following administration ofthis protein alone was completely unexpected because infection withrotavirus was not involved. Characterization of the parameters of thesenew models of rotavirus-induced diarrhea demonstrates that this entericviral-encoded protein is an enterotoxin, similar to bacterialenterotoxins which are wellknown to induce diarrhea by stimulatingsignal transduction pathways following interaction with specificintestinal receptors. The ordinary practitioner will appreciate thatthese new findings on NSP4-induced diarrheal disease and the datapresented herein support several novel therapeutic and preventiveapproaches to rotavirus-induced disease.

It is also reported here that a synthetic peptide corresponding to aa114-135 of SA11 NSP4 also induces an age-dependent diarrhea in youngmice comparable to NSP4 when administered by the IP and IL route. Sincethe NSP4 114-135 peptide was readily available in large amounts in pureform, we studied the response to the peptide in detail. The response tothe peptide was specific as shown by 1) lack of response to controlpeptides, 2) blocking with peptide-specific antibody, and 3) a mutatedpeptide (differing by only a single residue) alone failed to induce theresponse. The concentration of peptide required for disease inductionwas considerably higher than that needed for a response to the protein.Because the entire protein possesses more potent activity than thepeptide, other peptides from this protein which have the same effect arealso included in the present invention.

We have shown an analogous age dependence in the induction of diarrheawith purified NSP4 protein and NSP4 114-135 peptide. Mice were mostsensitive to the effects of the protein or peptide at 6-7 days of age.Diarrhea induction by NSP4 or NSP4 114-135 decreased as the age of theanimal increased, regardless of the route of administration. Hence theobserved diarrhea in this study mimics the properties of symptomaticinfection observed in experimental and natural rotavirus infection.

We have also shown that inoculation of NSP4 114-135 peptide-specificantiserum prior to IP delivery of peptide results in a dramaticreduction of disease. (90% reduction in disease).

We have also shown that diarrheal disease in pups born to dams immunizedwith the NSP4 114-135 peptide is significantly reduced in severity,duration, and in the number of pups with diarrhea.

We have also shown that post-infection administration of NSP4-specificantibody significantly reduces diarrheal disease.

These data, showing that NSP4 protein or NSP4 114-135-specificantibodies are sufficient to block the induction or severity ofdiarrhea, demonstrate that NSP4 and/or NSP4 114-135 and/or antibodiesspecific thereto will be useful as vaccines and as therapeutic agents.Additionally, new drugs can now be developed to block or minimize theeffects of interaction of NSP4 with its receptor or the effects of thedisruption of the calcium homeostasis in affected cells.

In addition, animals given peptide twice (at a two day interval) showeda rapid onset of severe diarrhea followed by stunted growth. The weightof these animals was 20-30% lower for three weeks after administrationof peptide.

Based on our results on NSP4-induced diarrhea in mice and rats, we haveproposed a model in which two intestinal receptors are required forsymptomatic rotavirus infection. One receptor binds rotavirus particlesresulting in virus entry and gene expression, but not necessarilydisease, whereas the second receptor is NSP4-specific. Additionally,while the receptor for rotavirus infection is maintained with age,allowing the adult mouse (34-35 days old) to replicate and excretevirus, the NSP4 fully functional receptor is not maintained withdevelopmental aging, so disease is not observed. In addition, disease isnot seen in adolescent mice (17-18 days old) because the colon of theseanimals can absorb fluid secreted in the small intestine.

We have shown that NSP4 114-135 promotes and augments cAMP-dependent Cl⁻secretion in mouse intestinal mucosa and induces diarrhea in rodents ina time frame similar to ST_(B) (about 3 hrs). The electrophysiologicaldata show that NSP4 induces calcium increases in the intestines of micein an age-dependent manner and these increases in calcium result inchloride secretion as measured by short-circuit currents. Directaddition of cross-linked NSP4 114-135 to mouse ileal mucosal sheetsresulted in a rise in current, similar to that evoked by the calciumagonist, carbachol. In addition to the age-dependence, induction ofchloride secretion from intestinal mucosal sheets was site-dependent.Zero to minimal responses were observed when mouse jejunum, duodenum orcolon tissue was employed, and maximum responses were induced when theileum was utilized. These results support our model of NSP4-induceddiarrhea.

Our data show that NSP4 stimulation of a Ca²⁺-dependent signaltransduction pathway, resulting in disruption of normal intestinalepithelial transport, is similar to that reported for guanylin and theheat-stable enterotoxins. Based on the enteropathogenic similarities inintestinal secretion with those reported for guanylin and theheat-stable enterotoxins, NSP4 can be considered a viral enterotoxin.

We have also shown that administration of virus or peptide to 5-7 dayold CFTR knock-out mice—mice homozygous for the mutation in the CFTRchloride channel coding region that causes Cystic Fibrosis—results indiarrhea in 100% of the cases.

We have also shown that administration of HIV gp160 to 6-7 day oldBalb/C mice causes diarrhea in 100% of the cases.

In accordance with the foregoing and with the disclosure that follows,it is an object of the present invention to provide a method for thescreening and identification of viral enterotoxins associated withrotavirus and other gastroenteritis viruses, such as caliciviruses,astroviruses, enteric adenoviruses, coronaviruses, and parvoviruses,including administering expressed proteins or peptides or syntheticpeptides of such viruses to animals and monitoring the animals fordiarrhea. For the purpose of this and other objects of the invention,human volunteers shall be considered to be within the scope of“animals.” It is a further object of the present invention to providemethods for the screening and identification of new viral enterotoxinsincluding administration of expressed proteins or peptides or syntheticpeptides to CD1 mice, Balb/C mice and/or Sprague-Dawley rats andmonitoring for diarrhea.

To the extent that the meaning of the term “diarrhea-genic viralprotein” may be construed to differ from the meaning of the term viralenterotoxin, if the term “diarrhea-genic viral protein” is substitutedfor the term viral enterotoxin, the subject matter of this and allfollowing objects and all claims is also considered to be within thescope of the invention, and fully described for all purposes.

It is another object of the present invention to provide methods for thescreening for and identification of viral enterotoxins associated withrotavirus and other gastroenteritis viruses, such as caliciviruses,astroviruses, enteric adenoviruses, coronoviruses and parvoviruses,including in vitro administration of virus, viral proteins or peptidesthereof to intestinal mucosa tissues or to cells and monitoring chloridesecretion and/or intracellular calcium levels and/or cAMP levels.

It is another object of the present invention to provide a method forthe screening for and identification of viral enterotoxins associatedwith viruses that are not known as diarrhea or gastroenteritis virus,but that are associated with diarrhea as a consequence of infection.Without limiting the invention, examples include human immunodeficiencyvirus (HIV) and cytomegalovirus (CMV). This method includesadministering expressed proteins or peptides or synthetic peptides of aselected virus to animals and monitoring the animals for diarrhea.

It is another object of the present invention to provide methods for thescreening for and identification of viral enterotoxins associated withother viruses associated with diarrhea, including HIV and CMV, includingin vitro administration of virus, viral proteins or peptides thereof tointestinal mucosa tissues or to cells and monitoring chloride secretionand/or intracellular calcium levels and/or cAMP levels.

It is another object of the present invention to provide a method fortreatment of diarrheal disease, including reducing the severity ofdiarrhea, caused by viral infection, including administering antibodiesto viral enterotoxins to a subject with diarrhea or known, or suspectedto be infected by or to have been exposed to a gastroenteritis virus.For the purpose of this invention, antibodies shall mean polyclonal andmonoclonal antibodies unless otherwise indicated. Methods for thepreparation of polyclonal and monoclonal antibodies to any protein orpeptide are well known to the practitioner having ordinary skill in theart.

It is another object of the present invention to provide a method forthe prevention or amelioration of diarrhea caused by rotavirus infectionincluding administration of antibodies to NSP4 protein or peptidesthereof, including but not limited to NSP4 114-135 and NSP4 120-147. Asrotavirus infection is transmitted rapidly, this method is considered toinclude the prevention or amelioration of disease following exposure toa known infected person, for example in day care centers and inhospitals.

For the purpose of this invention, the term “compound comprising aminoacids in a sequence corresponding to NSP4 114-135” shall mean a compoundwhich has within it a sequence of amino acids corresponding to thesequence of NSP4 114-135, including NSP4 114-135 and the NSP4 protein.For the purpose of this invention, the term “compound comprising aminoacids in a sequence corresponding to NSP4 120-147” shall mean a compoundwhich has within it a sequence of amino acids corresponding to thesequence of NSP4 120-147, including NSP4 120-147 and the NSP4 protein.For the purpose of this invention, the term “derivative” shall mean anymolecules which are within the skill of the ordinary practitioner tomake and use, which are made by derivatizing the subject compound, andwhich do not destroy the activity of the derivatized compound. Compoundswhich meet the foregoing criteria which diminish, but do not destroy,the activity of the derivatized compound are considered to be within thescope of the term “derivative.” Thus, according to the invention, aderivative of a compound comprising amino acids in a sequencecorresponding to the sequence of NSP4 114-135 or NSP4 120-147, need notcomprise a sequence of amino acids that corresponds exactly to thesequence of NSP4 114-135 or NSP4 120-147, so long as it retains ameasurable amount of the activity of the NSP4 114-135 or NSP4 120-147peptide.

It is another object of the present invention to provide monoclonalantibodies to NSP4 protein, to NSP4 114-135 peptide, to NSP4 120-147peptide, and to other peptides of NSP4.

It is another object of the present invention to provide a method forthe prevention of decreased growth rates caused by rotavirus infectionincluding use of the NSP4 protein or peptides thereof, including but notlimited to NSP4 114-135 and/or NSP4 120-147, as a treatment for orvaccine against rotavirus diarrhea.

It is another object of the present invention to use the NSP4 protein orpeptides thereof, including but not limited to NSP4 114-135 and NSP4120-147, to identify and/or characterize a new intestinal receptor whosesignaling induces secretion.

It is another object of the present invention to provide methods for theidentification and use of compounds, such as small molecule inhibitors,to bind the active domain of NSP4 or other viral enterotoxins toprevent, ameliorate or stop diarrheal disease. For the purpose of thisinvention, small molecule inhibitors shall mean any ligand that can bindwith high affinity to a target molecule, thereby inhibiting the targetmolecule's activity. Small molecule inhibitors include, but are notlimited to, peptides, oligonucleotides, amino acids, derivatized aminoacids, carbohydrates, and organic and inorganic chemicals. Libraries ofsmall molecule inhibitors are available to the practitioner eitheraccording to known methods, or commercially. Accordingly, this methodincludes identifying a viral enterotoxin, screening the purifiedenterotoxin against one or more random small molecule libraries, forexample, a random peptide library, a random oligonucleotide library, ora pharmaceutical drug library, and identifying those small moleculesthat bind with high affinity to the viral enterotoxin.

Another method for identifying small molecule inhibitors includes thesteps of identifying viral enterotoxins, determining the high resolutionstructure of these proteins and/or peptides thereof, determining theactive domain(s) and designing small molecule inhibitors which bind withhigh affinity to the active domain(s). Another method includesidentifying viral enterotoxins, identifying the intestinal receptorwhich binds the viral enterotoxin, and designing small moleculeinhibitors which competitively bind the receptor, without inducingsecretion.

It is another object of the present invention to provide a method forthe design of new drugs for the prevention of diarrhea and/or Ca²⁺mediated intestinal secretion including identifying the intracellularpathway by which [Ca²⁺]_(i) is increased and making compounds whichinhibit any step in the pathway. Specifically, this method includesidentifying the molecules active in the signalling pathway andidentifying compounds which inhibit their activity. Such compounds willinclude but not be limited to small molecule inhibitors which blockbinding of NSP4 to its receptor, blocking of G protein mediated or othersignal transduction secondary messengers and pathways which lead tochloride secretion or diarrhea.

It is another object of the present invention to provide a method forthe diagnosis of rotavirus infection including the detection of NSP4 instools of individuals with diarrhea. Detection of peptides of NSP4 isconsidered to fall within the scope of detection of NSP4.

It is another object of the present invention to provide a method forthe diagnosis of rotavirus infection including the detection ofantibodies to NSP4 in the sera or stools of individuals with diarrhea.

It is another object of the present invention to provide a vaccinecomprising the NSP4 protein or peptides thereof, including but notlimited to NSP4 114-135 and NSP4 120-147, to induce the formation ofprotective active or passive antibodies.

It is another object of the present invention to provide a vaccinecomprising a toxoid form of the NSP4 protein, including but not limitedto formaldehyde inactivated NSP4 to induce the formation of a protectiveimmune response.

It is another object of the present invention to provide vaccinesagainst gastroenteritis viruses, including rotaviruses, caliciviruses,astroviruses, enteric adenoviruses, coronoviruses and parvoviruses,including viral enterotoxins which induce the diarrhea associated withviral infection.

It is another object of the present invention to provide methods for theidentification of potential vaccines against gastroenteritis viruses,including screening for viral enterotoxins, raising antibodies againstany identified possible enterotoxins, and determining whether theantibodies protect against disease caused by the virus.

It is another object of the present invention to provide a method tomonitor vaccine efficacy or protective immunity by determining theimmune response to NSP4 protein and/or to peptides thereof.

It is another object of the present invention to provide a method forimmunization against rotavirus infection comprising administering to asubject a vaccine including the NSP4 protein or peptides thereof,including but not limited to NSP4 114-135 and NSP4 120-147 peptides.

It is another object of the present invention to provide a method forimmunization against rotavirus infection comprising administering to asubject a vaccine comprising a toxoid form of the NSP4 protein.

It is another object of the present invention to identify key residuesin NSP4 responsible for its ability to induce diarrhea and thus toidentify specific amino acid sequences associated with avirulence.Having done this, gene 10 from these avirulent viruses may be used toselect and produce reassortment viruses that contain a gene 10 thatconfers an avirulent phenotype which can be used as live attenuatedreassortment virus vaccine candidates.

It is another object of the present invention to provide a method ofidentifying a virulent strain of rotavirus by determining the amino acidsequence of the NSP4 protein of the strain.

It is another object of the present invention to provide a method ofpassive immunization against rotavirus infection including administeringto an expectant mother a vaccine including the NSP4 protein or peptidesthereof, including but not limited to NSP4 114-135 and NSP4 120-147.

It is another object of the present invention to provide a method forthe intentional induction of intestinal secretion includingadministration of the NSP4 protein or peptides thereof, including butnot limited to NSP4 114-135 and NSP4 120-147.

It is another object of the present invention to provide a method forthe treatment of cystic fibrosis including administration of NSP4protein or peptides thereof, including but not limited to NSP4 114-135and NSP4 120-147, to enhance fluid secretion.

It is another object of the present invention to provide a method forthe treatment of cystic fibrosis comprising administering NSP4 orderivatives or new molecules that act like NSP4 to enhance secretionthrough the same mechanism NSP4 uses.

It is another object of the present invention to provide a new laxativeincluding the NSP4 protein or peptides thereof, including but notlimited to NSP4 114-135 and NSP4 120-147.

It is another object of the present invention to provide methods for thescreening of antiviral compounds, compositions and/or treatments and/orthe evaluation of vaccine efficacy, including administering viruses,viral proteins or peptides thereof to one or more of three new animalmodels for diarrheal virus infections, the CD1 mouse, the Balb/C mouseand the Sprague-Dawley rat.

DESCRIPTION OF THE DRAWINGS

FIG. 1A Rotavirus NSP4 protein induces diarrhea in CD1 mice. 0.1 to 5nmols (2-100 μM) of purified NSP4 was administered by the IP or ILroutes to 6-7, 8-9 and 17-18 day old CD1 pups. Rotavirus protein VP6 wasused as the control in 6-7 day old animals, the most sensitive. The doseand route of the proteins, age of the animals, and mean diarrhea score(mean score) are indicated on the bottom of the graph. The Y axisdisplays the % diarrhea. Above each column is the number of responders(mice with diarrheal disease) over the total number of animals tested.

FIG. 1B IP administration of NSP4 induces diarrhea in 6-7 dav old CD-1pups. 0.04 to 1.0 nmols (1-25 μg) of purified NSP4 protein wasadministered to 6-7 day old CD1 pups by the IP route. The doseadministered, in nanomoles and in micrograms, is shown on the X-axis.The percentage of pups that display diarrhea in response to theadministered dose is shown on the Y-axis. Above each column the numberof responders (pups with diarrhea) over the total number of animalsreceiving treatment is shown.

FIG. 2A Diarrheal response in CD1 mice following IP administration ofNSP4 114-135 peptide. Young (6-7 day) mouse pups were injected withvarious doses of peptide (x axis) and monitored for disease. The numberof responders over the total number animals tested is shown above eachcolumn. With the CD1 mice, 0.1-50 nmol (2 μM-1 mM) of peptide elicitedsimilar responses (30-40% diarrhea induction); and 100-400 nmols (2-8mM) of peptide elicited comparative responses with 60-70% of the animalssick. A slight increase in disease was noted with 500 nmol (10 mM) ofpeptide. No diarrhea was induced with 0.001 nmol of peptide (data notshown).

FIG. 2B Diarrheal response in Balb/C mice following IP administration ofNSP4 114-135 peptide. Young (6-7 day) mouse pups were injected withvarious doses of peptide (x axis) and monitored for disease. The numberof responders over the total number animals tested is shown above eachcolumn. With the Balb/C pups, diarrhea induction was seen in 100% of theanimals with 50 nmol (1 mM) of peptide indicating the inbred animals aremore sensitive to the effect of the NSP4 peptide.

FIG. 3A IP delivers of NSP4 114-135 peptide induce an age-dependentdiarrhea in CD1 mice and Sprague-Dawley Rats. Different age outbred miceand rats were inoculated (IP) with NSP4 114-135 peptide and evaluatedfor disease. The age and species of the pups, dose of the syntheticpeptide and indication of whether peptides were unlinked or cross-linkedare indicated on the bottom of the graph. The dose of the IP deliveredpeptide varied with the age of the animals, i.e., older animals receiveda higher dose to control for the differences in body weight. The Y axisindicates the % diarrhea and above each column is indicated the numberof responders over the total number of animals inoculated. The peptidewas diluted in sterile PBS and evaluated for sterility. A final volumeof 50 μl per dose was used. Analogous to the effects of purified NSP4,additional symptoms included lethargy and coldness to the touch.

FIG. 3B IL delivery of NSP4 114-135 peptide induce an age-dependentdiarrhea in CD1 mice and Spraque-Dawley Rats. Different age outbred miceand rats were inoculated (IL) with NSP4 114-135 peptide and evaluatedfor disease. The age and species of the pups, dose of the syntheticpeptide and indication of whether peptides were unlinked or cross-linkedare indicated on the bottom of the graph. The Y axis indicates the %diarrhea and above each column is indicated the number of respondersover the total number of animals inoculated. The peptide was diluted insterile PBS and evaluated for sterility. A final volume of 50 μl perdose was used. Analogous to the effects of purified NSP4, additionalsymptoms included lethargy and coldness to the touch.

FIG. 4 Dose Response for Cross-Linked NSP4 Peptide Delivered IP to 6-7day old CD1 and Balb/C Mice. The NSP4 114-135 peptide was cross-linkedto itself with glutaraldehyde and dialyzed against sterile PBS prior toIP delivery to CD1 and Balb/C mice. The number of responders over thetotal number of animals inoculated is shown above each column. Thedisease response to the cross-linked peptide was seen at lower doseswhen compared to the peptide alone. Additional symptoms includedlethargy and coldness to the touch.

FIG. 5 illustrates the experimental designs used to test the ability ofNSP4 114-135 to induce protective immunity from infectious rotaviruschallenge and to test the ability of NSP4-specific antibody to mitigaterotavirus diarrhea following infection. The left hand side of the figureillustrates that mouse dams were immunized with NSP4 peptide or withcontrol peptide and then bred. Pups born to the dams were orallychallenged with virulent rotavirus at 6-7 days. The right hand side ofthe figure illustrates how pups born to non-immunized dams were firstorally challenged with virulent rotavirus, followed by oral gavage ofNSP4-specific or control antisera. The results of these experiments areset forth in Tables 5 and 6.

FIG. 6 illustrates the results from an experiment to study thedifferences in weight and growth between normal animals and animalssuffering from NSP4 114-135-induced diarrhea.

FIG. 7 Amino acid sequence comparison of NSP4 from OSU attenuated andOSU virulent virus. The amino acid sequence of the NSP4 protein of OSU-a(a porcine rotavirus, tissue culture attenuated, avirulent strain), topline, is compared to the amino acid sequence of the NSP4 protein ofOSU-v (a porcine rotavirus, virulent strain), bottom line. Positions atwhich the two sequences are different are shown in bold.

DETAILED DESCRIPTION OF THE INVENTION

Materials and Methods

EXAMPLE 1

NSP4.

NSP4 was purified from recombinant-baculovirus pAC461-G10 infectedSpodoptera frugiperda (Sf9) cells expressing gene 10 by FPLC on a QMAanion exchange column as previously described (15, 16), and with anadditional affinity purification step on a column containing anti-NSP4antibodies. Different NSP4 preparations of ≧70% and 90% purity gave thesame biologic results. The protein was sterile based on bacteriologicculturing in L-broth incubated at 37° C. for one week, and lackedendotoxin based on testing by the limulus amebocyte lysate (LAL) assay(17). VP6 was purified to >95% purity from recombinant-baculoviruspAc461/SA11-G6 infected Sf9 cells by gradient centrifugation aspreviously described (18). Both proteins were lyophilized and diluted insterile PBS to a final volume of 50 μl per dose, regardless of the routeof administration.

EXAMPLE 2

Synthetic Peptides.

Synthetic NSP4-specific and control peptides utilized in this study wereoriginally selected based on algorithms which predict surface potential(19), turn potential (Pt) (20), and amphipathic structure (21). A blocklength of 11 was used and an amphipathic score (AS) of 4 was consideredsignificant. We selected sequences with unusually high predictedpropensities for folding into amphipathic helices and reverse turns,because small peptides which typically lack any folding pattern in anaqueous environment can fold into an ordered secondary structureresembling the nascent protein if the structural propensity is high(22).

Peptide sequences used in this study include: NSP4 114-135 (23),(DKLTTREIEQ VELLKRIYDKLT, SEQ ID NO. 1), AS=35; a peptide from theamino-terminus of NSP4, NSP4 2-22 (EKLTDLNYTLSVITLMNNTLH, SEQ ID NO. 2),AS=14; an extended highly amphipathic peptide, NSP4 90-123(TKDEIEKQMDRVV KEMRRQLEMIDKLTTREIEQ, SEQ ID NO. 3) AS=71; a mutated NSP4114-135 peptide, mNSP4 131K (DKLTTREIEQVELLKRIKDKLT, SEQ ID NO. 4)AS=31; and a peptide from the COOH— terminus of the Norwalk virus capsidprotein having a centrally located tyrosine residue (24), NV 464-483(DTGRNLGEFKAYPDGFLTCV, SEQ ID NO. 5) AS=41 (table 1), and NSP4 120-147(EIEQVELLKRIYDKLTVQTTGEIDMTKE, SEQ ID NO.6) AS=35.0.

All peptides were synthesized by the University of Pittsburgh PeptideCore Facility employing Fmoc chemical strategy and standard protocols(25). Coupling and deblocking efficiencies were monitored by theninhydrin colorometric reaction (26). Peptides were cleaved from theirsolid resin support and separated from organic contaminants by multiplecold ether extractions, and conventional gel filtration chromatography(Sephadex G-25). The final peptide product was characterized byreverse-phase HPLC (Deltapak C4, Waters) and plasma desorption massspectroscopy (27). Only those peptides with the correct theoretical massand 90% or greater full-length product were employed in these studies.Prior to use, peptides were further purified either by HPLC on asemi-preparative, reverse-phase C18 column (Bondapak, Waters) or bymultiple elutions from a conventional gel filtration column (1.5 mm×40mm). Peptide purity was confirmed prior to inoculations by gelfiltration chromatography (Protein-Pak 60 column, 10 μm, Waters) on aWaters HPLC unit. The elution profiles were monitored by UV absorption(Lambda-Max LC-spectrophotometer, Waters) at 220 nm and recorded by a745 Data Module (Waters). The elution buffer was PBS, pH 7.2, and theflow rate 0.5 ml/min. Sterility was confirmed as described for NSP4protein.

EXAMPLE 3

Glutaraldehyde Cross-linking of Synthetic Peptides.

In some cases, peptides were cross-linked to themselves or to thecarrier protein, keyhole limpet hemocyanin (KLH), by glutaraldehyde in asingle-step coupling protocol (28). Briefly, the peptide immunogen wascoupled to KLH at a ratio of 100 nmol peptide: 1 nmol KLH or to itselfat a 1:1 ratio by the addition of glutaraldehyde to a finalconcentration of 0.4%. The reaction was quenched by the addition of 1Mglycine (C_(t)=20 mM). The cross-linked peptides were extensivelydialyzed against sterile PBS prior to use.

EXAMPLE 4

Antibody Production.

NSP4 114-135 peptide-specific antiserum was generated in CD1 mice andNew Zealand white rabbits by immunization with peptide cross-linked viaglutaraldehyde to the protein carrier KLH, as described above. The firstinoculum was emulsified in Freund's complete adjuvant(Gibco), whereasall subsequent inoculations were prepared in incomplete Freundsadjuvant. Rabbits were injected intramuscularly (IM, once in each hip)and subcutaneously (SC) across the back of the neck. Boosting doses ofemulsified antigen (100 nmol of peptide) were done every 4 wk. for atotal of 5 immunizations. Mice were immunized every three weeks by theIM, SC and IP routes. Preimmunization and postimmunization sera wereevaluated by peptide ELISAs (titer of 400-3200) as previously described(29) and by Western blot analyses.

EXAMPLE 5

IP and IL Administration of Protein and Peptides.

Purified NSP4 protein, peptide alone, or cross-linked to itself, wereadministered to young (6-10 days) and older (11-25 days) outbred CD1 orinbred Balb/C mice, and outbred Sprague-Dawley rats by theintraperitoneal (IP), intraileal (IL), intramuscular (IM), subcutaneousand oral routes. The peptide or protein inocula were diluted in sterilePBS to a final volume of 50 μl per does, regardless of the route ofadministration or inoculum. A 30 G needle was employed for the IP and ILdelivery of the inocula. Peptide was delivered orally to young mice bygavage using a PE-10 polyethylene flexible tubing (Intramedic, BectonDickinson) and food coloring. For the surgical introduction of thepeptide or protein via the IL route, animals were anesthetized withisofurane (Anaquest), a small incision was made below the stomach, theinocula were directly injected into the upper ileum, and the incisionwas sealed with polypropylene sutures (PROLENE 6-0, Ethicon). The pupswere isolated, kept warm, and closely monitored for a minimum of 2 hoursprior to returning them to their cage.

EXAMPLE 6

Monitoring of Diarrhea Induction.

Diarrhea induction by the NSP4 protein and peptides was carefullymonitored for 24 hours following the inoculations. Each pup was examinedevery 1-2 hr for the first 8 hr and at 24 hr post inoculation by gentlypressing on the abdomen. Diarrhea was noted and scored from 1 to 4 witha score of 1 reflecting unusually soft, loose, yellow stool, and a scoreof 4 being completely liquid stool. A score of 2 (mucous with liquidstool, some loose but solid stool) and above was considered diarrhea. Ascore of 1 was noted, but was not considered as diarrhea. The scoringwas done by a single person and the pups were coded during analysis ofdiarrhea. Other symptoms monitored included lethargy, coldness to thetouch, and ruffled coats in older animals.

EXAMPLE 7

Analysis of Chloride Secretion Reonsiveness to NSP4 114-135 in theIntestinal Mucosa of Mice.

Unstripped intestinal mucosal sheets from 19-22 and 35 day old mice wereanalyzed for chloride secretory responsiveness to NSP4 114-135.Short-circuit currents (I_(ac)) were measured across unstrippedintestinal mucosal sheets from 19-22 and 35 day old CD1 mice using anautomatic voltage clamp (Bioengineering, Univ. of Iowa) as describedpreviously (30). The mid-ileum of the mouse intestines was utilized. Theunstripped mucosal sheets taken from the intestine were placed intomodified Using chambers with 0.12 cm² apertures (machine shope, UTHSC)and transepithelial potential (V_(t)) was registered by 3 M KCl agarbridges connected to balanced calomel half-cells. The transepithelialcurrent required to clamp V_(t) to 0 was passed through Ag—AgClelectrodes connected to the 3 M KCl bridges. All experiments wereperformed at 37° C. in bicarbonate Ringers solution gassed with 95%O₂-5% CO₂ by airlift circulators as previously described (same asabove). The mucosal bath contained sodium-free (N-methyl-D-glutamine)substituted Ringers to minimize the effects on I_(ac) of cAMP stimulatedelectrogenic Na⁺/glucose co-transport across the small bowel (31).Following temperature and ionic equilibration, basal I_(ac) measurementswere taken and intestinal mucosal sheets were challenged withcross-linked peptide (either NSP4 114-135, NSP4 2-22, or mNSP4 131K),the calcium-elevating agonist carbachol (Cch), or the cAMP-agonistforskolin (FSK). Bumetamide sensitivity was tested and confirmed thechloride secretory response.

Results

EXAMPLE 8

NSP4 Protein Induces Age-dependent Diarrhea in Mice.

Whether administration was IP or intraileal (IL), diarrhea was observedwithin 1 to 4 hr post inoculation, typically continued for up to 8 hr,but occasionally persisted for 24 hr. Purified NSP4 (0.1-5 nmol) wasadministered by the IP route to 6-7 and 8-9 day old CD1 pups. In 6-7 dayold CD1 pups, IP administration of 0.1 nmol of NSP4 induced diarrhea in60% of the mice, whereas no disease was induced in 8-9 day old mice withthe same concentration of protein (FIG. 1A). IP administration of 1 nmolof NSP4 resulted in 100% of the 6-7 day pups with diarrhea, and 60% ofthe 8-9 day old mice with disease. A larger dose of 5 nmol of NSP4induced diarrhea in 90% of the older (8-9 day) mice. Additional clinicalsymptoms included lethargy and coldness to the touch, which wereobserved in the majority of treated animals with diarrhea of all ages.The induction of diarrhea by NSP4 was shown to be specific for thisprotein as administration of the same volume of buffer or VP6 had noeffect.

IL administration of 0.5 nmol of purified NSP4 protein resulted indisease in 100% of the CD1 pups (8-9 day old mice) within the first 2 hrpost inoculation, whereas no diarrhea was observed in 17-18 day old pups(Table 2, FIG. 1).

Thus, the response to NSP4 was age- and dose-dependent in CD1 pups. Inaddition, the induction of diarrhea by NSP4 was specific, asadministration of the same concentration of purified rotavirus VP6 orthe same volume of buffer had no effect (FIG. 1). The effect of IP andIL delivery of NSP4 protein in mice is the same. Intramuscular (IM)inoculation of 1 nmol of purified NSP4 produced no ill effects (data notshown). Subcutaneous and oral administration of NSP4 also produces noill effects (data not shown).

Additional data showing a dose response in 6-7 day old CD1 pups ispresented in FIG. 1B. The amount of peptide administered is shown innanomoles and micrograms. 0.04-1.0 nmols (1-25 μg) of purified NSP4 wasadministered to 6-7 day old CD-1 pups by the IP route. A correlationbetween increasing incidence of diarrhea and increasing dose was seen(FIG. 1B) over the range tested. The highest tested dose (1.0 nmol=25μg) induced diarrhea in all mice tested (10 of 10).

EXAMPLE 9

NSP4 114-135 Peptide Induces Diarrhea in Mice.

The NSP4 114-135 peptide has an AS of 35, is localized in thecytoplasmic domain of NSP4, and mobilizes intracellular calcium ineukaryotic cells (15, 16).

Following IP administration of 0.1 to 50 nmol of the NSP4 114-135peptide, a similar disease response was noted in 6-7 day old CD1 outbredpups with 30-40% diarrhea induction (FIG. 2A). The percentage of CD1pups with diarrhea increased to 60-70% following the IP delivery of100-400 nmol of NSP4 114-135 and 89% of pups had diarrhea followingadministration of a dose of 500 nmol of peptide. Induction of disease in100% of the CD1 pups was not achieved; doses exceeding 500 nmol were notadministered since the volume of each dose was limited to 50μl . Thesedata indicate the disease response in CD1 mice can be divided into threegroups based on the dose of the NSP4 114-135 peptide, 1) less than andequal to 50 nmol (1 mM) resulting in 30-40% of the animals with disease,2) 100-400 nmol (2-8 mM) yielding disease in 60-70% of the animals, and3) 500 nmol and (10 mM) above inducing diarrhea in at least 89% of theyoung mice.

Diarrhea was induced in 100% of the 6-7 day old Balb/C pups with lowerconcentrations (only 50 nmol) of peptide (FIG. 2B), and diarrhea wasobserved in 80% of the Balb/C mice given 0.1 nmol (2μM) of NSP4 114-135.Hence the Balb/C pups appeared more sensitive to the effects of NSP4114-135.

Taken together, doses exceeding 50 nmol (1 mM) of NSP4 114-135 peptidewere sufficient to induce diarrhea in the majority of young mice whenadministered by the IP route. The diarrhea was observed within 1 to 4 hrpost inoculation and typically continued for up to 8 hr, butoccasionally was present for 24 hr. The severity of diarrhea typicallyincreased with time. That is, a mouse with a diarrhea score of 1 in thefirst hr post inoculation would have a diarrhea score of 4 in the nexthr. Various degrees of lethargy were noted following the administrationof peptide and this was most pronounced at 3 to 4 hr post inoculation.The lethargy was accompanied by the pups being cold to the touch and wasage-dependent. The severity of the induced diarrhea was greater in theBalb/C pups. No symptoms were noted with control peptides (NSP4 2-22, NVC-terminus) or PBS administered to the same age and species of mice.

EXAMPLE 10

NSP4 120-147 Peptide Induces Diarrhea in Mice.

A peptide corresponding to amino acid residues 120-147 of NSP4 wasprepared and tested in 5-7 day old pups. When a dose of 100 nmols wasadministered, all (5 of 5) animals exhibited severe diarrhea. A dose of5 nmols induced diarrhea in 7 out of 8 animals (88%). This demonstratesthat other peptides derived from NSP4 can be prepared and screened tofind the peptide with the highest activity. It is well within theability of one of ordinary skill in the art to synthesize and screen alibrary of overlapping peptides that represents the entire sequence ofthe NSP4 protein in order to locate peptides with biological activity.One skilled in the art can readily appreciate that both the length ofthe peptides, and the number of residues that overlap in adjacentpeptides, can be varied at the discretion of the practitioner withoutdeviating from the spirit of the present invention.

EXAMPLE 11

Diarrhea induction in CD1 and Balb/C Mice by Cross-linked NSP4 114-135.

The NSP4 114-135 peptide was cross-linked to itself by glutaraldehydeand administered to young mouse pups by the IP route to determine if thediarrhea induction was affected by structure or oligomerization.Diarrhea was induced in the majority of the CD1 pups at a lower dose ofNSP4 114-135 when the peptide was cross-linked to itself when comparedto the peptide alone (FIG. 4). One nmol of cross-linked peptide induceddiarrhea in 80% of the CD1 pups which increased to 90% with 250 nmol ofcross-linked NSP4 114-135. As illustrated in FIG. 3, doses at or above 1nmol (20 μM) of cross-linked peptide were sufficient to elicit aresponse in the majority of the CD1 pups. Increasing the dose above 1nmol of cross-linked NSP4 114-135 had little effect, indicating thediarrheal response could not be increased with increased amounts ofsynthetic peptide, or that the response, once stimulated, could besaturated or additional stimulation had no effect.

Similar to the response in CD1 mice, diarrhea induction in 100% of theBalb/C pups was achieved with a lower dose (10 nmol, 200 μM) ofcross-linked peptide when compared to the peptide alone (FIG. 4). Inaddition, the lethargy and coldness to the touch were more severe andlasted longer in animals that received the cross-linked peptide.Cross-linked NSP4 2-22 and NV C-terminus peptides were administered ascontrols and did not induce symptoms in young mice.

Induction of disease at a lower dose and with greater severity with theIP administration of cross-linked NSP4 114-135 suggests thatcross-linking either stabilizes the peptide, oligomerizes the peptide,or results in a conformation more closely resembling the native protein.These data suggest structure may be important for disease induction.

EXAMPLE 12

Cross-linked NSP4 114-135 Peptide also Induces Diarrhea in Young Rats.

The NSP4 114-135 peptide was tested in a second species, theSprague-Dawley rat to determine whether the disease response induced bythis peptide was only effective in young mice. IP inoculation of 100-250nmol of cross-linked peptide induced diarrhea in 78% of young (6 days)rat pups and in none of the older (10 day) rat pups (FIG. 3A). Nodisease was observed in the same age rodents administered controlpeptides. The response in rats was slower than that observed in mice,taking from 6 to 12 hr before the onset of diarrhea was noted, comparedto 2 to 4 hours post inoculation for the mice, and required a higherconcentration of peptide to observe disease. However, the induceddiarrhea and lethargy in the young rats frequently persisted for up to48 hr. These differences may reflect the difference in size andintestinal transit time between the rat and mouse or species (genetic)variation.

EXAMPLE 13

IL Delivery of NSP4 Peptide.

IL administration of 120-240 nmol of cross-linked NSP4 114-135 induceddiarrhea in 90% of young (6-7 days) rat pups. Analogous to the responseof young rats following the IP administration of cross-linked peptide,the onset of diarrhea was slower than that seen in the mice, taking from6 to 12 hr, but lasted for a greater length of time (up to 48 hr). Thesurgical introduction of 10 nmol (200 μM) of cross-clinked peptideinduced diarrhea in 100% of the young (8-9 days) Balb/C pups, identicalto the induction of diarrhea following IP delivery (Table 2). Theage-dependence of the diarrhea response noted with the IP administrationof cross-linked NSP4 114-135 was maintained with the IL administrationof cross-linked peptide. Only one-third of the 11-12 day old Balb/C micehad diarrhea when administered 10 nmol of cross-linked peptide by the ILroute, and none of the 15-17 day animals had diarrhea. In addition,older CD1 mice (11-12 and 25 days) had no ill effects from the ILdelivery of 50-200 nmol (1-4 mM) of cross-linked peptide (FIG. 3B, Table2). An equal concentration of cross-linked NSP4 2-22 peptide or an equalvolume of PBS, when surgically introduced in both young and olderrodents, had no ill effects (data not shown).

Hence, the effect of IP and IL delivery of NSP4 peptide in rodents wasequivalent.

EXAMPLE 14

Diarrhea Induction is Age Dependent.

Since rotavirus-induced diarrhea is age-dependent, we tested thisparameter with the peptide. Between 100 and 300 nmol of NSP4 114-135peptide, alone or cross-linked, was administered by the IP route todifferent age outbred mice and rats. Diarrhea was observed in the youngmice within 2 to 4 hr post inoculation, whereas reduced or no symptomswere seen in older (11-12 or 15-17 days) animals (FIG. 3). With IPadministration of peptide alone, disease was induced in 60% of the 6-7day old CD1 pups with no symptoms noted in the 11-12 and 15-17 day oldmice. IP administration of cross-linked peptide resulted in 90% diarrheainduction in 6-7 day old CD1 pups, 30% disease in 11-12 day old pups,and only 10% disease in 15-17 day old mice.

A comparable age dependence was observed with the Sprague-Dawley ratswhen cross-linked peptide was administered by the IP route. Diarrhea wasdetected 6 to 12 hr post inoculation in 78% of the young (5-6 day) ratswhile no disease was seen in the 10 day old rats given a similar dose ofcross-linked peptide (FIG. 3). Thus an age dependence, similar to whatis seen in a natural infection, is seen with the NSP4 114-135 peptide.

EXAMPLE 15

Induction of Diarrhea is Dose Dependent

To determine if the response to the NSP4 114-135 peptide wasdose-dependent, 0.1-500 nmol of peptide were administered IP to 84 CD1pups (6-7 days old; FIG. 2). The disease response to the NSP4 114-135peptide was dose-dependent (χ² _(trend)=9.98, p=0.0016) with a DD₅₀ (50%diarrheal dose) of 79 nmol (10).

EXAMPLE 16

Specificity of the Diarrhea Response to Peptide NSP4 114-135.

Specificity of the diarrhea induction by the NSP4 114-135 peptide wasconfirmed by the administration of a panel of control peptides to youngmouse pups (Tables 1 and 3).

Mutant peptide mNSP4 131K, in which the tyrosine at position 131 of NSP4114-135 is replace with a lysine did not induce diarrhea (0/11),indicating the importance of this tyrosine residue in the induction ofdiarrhea. Neither did NSP4 2-22 or NV 464-483 cause diarrhea, 0/11 and0/10, respectively. NSP4 90-123, which overlaps the 114-135 peptide by 9residues, induced diarrhea in only 20% (2/10) of the mice tested (Table3). The percentage of diarrhea induction increased to 50% when the NSP490-123 peptide was crosslinked. Cross-linked mutant (m)NSP4 131k peptideinduced diarrhea in 2 of 10 mice, while cross-linked NV 464-483 did notcause disease. Thus, the response to peptide alone appears to bedirected to a region of NSP4 inclusive of residues 114-135.

EXAMPLE 17

Administration of Peptide NSP4 114-135 Results in Stunted Growth.

Animals given peptide three times per day for two days showed a rapidonset of severe diarrhea followed by stunted growth. The weight of theseanimals was 20-30% lower for three weeks after administration ofpeptide, (FIG. 6). These results mimic characteristics of rotavirusdisease in animals and children, including the fact that both may showdecreased growth rates after multiple infections.

EXAMPLE 18

Antiserum to NSP4 114-135 Peptide Blocks Induction of Diarrhea

We also evaluated whether antiserum made to the NSP4 114-135 peptide wasable to block the induction of diarrhea (32). In the absence ofantibody, IP delivery of 50-100 nmol of NSP4 114-135 peptide induceddiarrhea in 67% of the mice. IP inoculation of NSP4 114-135peptide-specific antiserum 5 mins prior to IP delivery of peptide(50-100 nmol) resulted in a 90% reduction of disease. IP administrationof normal rabbit serum prior to peptide did not block the diarrhea.

EXAMPLE 19

NSP4 Antibodies Protect Against Virus-induced Disease.

The potential of NSP4 antibodies to protect against virus-induceddisease was tested by challenging pups, born to dams which wereimmunized with the NSP4 114-135 peptide or a control peptide, with ahigh dose of infectious SA11 virus (33), FIG. 5, left hand side.Diarrheal disease in pups born to dams immunized with the NSP4 114-135peptide was significantly (Fisher's exact test) reduced in severity,duration, and in the number of pups with diarrhea (Table 5). The NSP42-22 peptide was used as a control peptide, as it does not inducediarrhea in pups.

In another experiment, young mouse pups were infected with SA11 virus,and NSP4 antiserum or control antiserum was orally administered every4-6 hours for 60 hr, FIG. 5, right hand side. The pups administeredNSP4-specific antibody had significantly reduced diarrheal diseasecompared to animals given no treatment, rabbit pre-immune serum ornormal rabbit serum (NRS) (33), (Table 6). These data show the potentialof NSP4 antibodies to block rotavirus-induced disease.

EXAMPLE 20

Electrophysiological Analyses

The above data suggest NSP4 causes diarrhea by acting as an enterotoxin.Because enterotoxins stimulate net secretion in ligated intestinalsegments without histological alterations, or stimulate secretion inUsing chambers, the effects of the peptide, and known Ca²⁺—andcAMP-elevating agonists were tested on unstripped mouse intestinalmucosal sheets in modified Using chambers (14). Addition of forskolin(FSK, cAMP agonist) and carbachol (Cch, cholinergic agonist whichmobilizes Ca²⁺) to normal mouse ileal mucosa resulted in measurableelevations in Cl⁻ secretory short circuit current (I_(ac) Table 4).Addition of either 5 μM of NSP4 114-135 peptide (cross-linked to itselffor enhanced stability) or 5 μM of Cch to mucosal sheets of 19-22 dayold CD1 mice induced small (3 or 9 μA/cm², respectively) and transient(1-2 min) increases in I_(ac). When the mucosal sheets were exposed to 5μM of the cAMP-mobilizing agonist, FSK, larger increases in I_(ac) (44μA/cm²) were elicited that reached sustained levels within 2-3 min.After FSK pretreatment, challenge of the mucosa with either peptide orCch resulted in much larger increases in mucosal I_(ac) (64 or 63μA/cm², respectively) both the peptide and Cch potentiated the responseto FSK. All of the responses to agonists were sensitive to bumetamideand treatment of ileal mucosal sheets with cross-linked control NSP42-22 peptide did not induce a response. Addition of Cch to 19-22 day oldmouse mucosal sheets which had been pretreated with peptide alone, orpeptide in combination with FSK, had minimal or no additional effect onI_(ac). This subsequent loss of sensitivity to the Ca²⁺-elevatingagonist (Cch) after peptide pretreatment suggests that the NSP4 peptideincreases I_(ac) through changes in intracellular Ca²⁺ ([Ca²⁺]i).Addition of Cch to mucosa from a 35 day old mouse again elicited a small(14 μA/cm²) and transient (1-2 min) response that potentiated the effectof FSK (64 μA/cm²), whereas there was no or minimal increase in I_(ac)when the NSP4 114-135 peptide was added alone or with FSK to the 35 dayold mouse mucosal sheets (Table 4).

The electrophysiological responses from 19 day old mice initially seemparadoxical to the biological data since measurable secretion was notobserved as diarrhea in this age animal. Diarrhea likely was not seen inthese older animals because of fluid reabsorption by the colon. Thishypothesis was tested by IL administration of 200 nmol of NSP4 114-135or control peptide to 19 day old pups. At 4 hrs post inoculation, themice were sacrificed and the intestines were tied off, removed, weighed,and the length measured. The pups given NSP4 114-135 peptide showedsignificant fluid accumulation when compared to the control pupsalthough no diarrhea was seen in any animals.

We anticipate that younger mice would show a greater increase in I_(ac)than that seen in the 19 day old mucosa. However, intestinal mucosa fromyounger mice (<19 days) could not be mounted efficiently into the Usingchambers due to their small size; such experiments in very young micewill require the development of new methods to measure Cl⁻ secretion invitro. Nonetheless, the NSP4 114-135 peptide did not augment secretionin 35 day old mice, correlating the age-dependence seen in vivo.

EXAMPLE 21

Model for Rotavirus-induced Diarrheal Disease.

Based on our results on NSP4-induced diarrhea in mice and rats, wepropose a model in which two intestinal receptors are required forsymptomatic rotavirus infection. One receptor binds rotavirus particlesresulting in virus entry and gene expression, but not necessarilydisease, whereas the second receptor is NSP4-specific. NSP4 expressed ininfected cells would be released into the lumen and interact with thesecond receptor on adjacent cells. This interaction would trigger asignal transduction pathway, thereby increasing [Ca²⁺]_(i) levels andaugmenting endogenous intestinal secretory pathways. Using a newlyestablished ELISA which is sensitive enough to detect 31.3 ng or 0.02nmol of NSP4, we have detected NSP4 in the diarrheal stools of rotavirusinfected mice at concentrations necessary to induce disease. NSP4 wasnot present in stools from animals without diarrhea.

This model fits available data on rotavirus-induced diarrhea. In youngmice, homologous and heterologous rotaviruses cause diarrheal disease.For example, in young mice infected with the simian virus, SA11,infectious virus is not produced, histopathologic blunting of the villiis not observed, but diarrhea is induced (34). In other animals,diarrhea is seen prior to histologic changes (12). Adult mice arereadily infected by murine rotaviruses, but do not display diarrhea orother symptoms (35). However, virus can be isolated from fecal samplesand virus replication can be demonstrated in intestinal cells from adultanimals (32).

According to this model, the intestines of young mice possess aNSP4-specific receptor that decreases in number or structure or activityas the mouse ages, and interactions with this receptor stimulate Cl⁻secretion resulting in the observed diarrheal disease. Our modelpredicts that the binding activities or concentration of NSP4 receptorsis significantly reduced in adult animals such that the colon canaccommodate the increase in fluid secretion. The adult mouse canreplicate and excrete virus, but no disease is observed. That is, whilethe receptor for rotavirus infection is maintained with age, allowingthe adult mouse to replicate and excrete virus, the NSP4 receptor is notmaintained with developmental aging, so disease is not observed.

Further support for our model comes from our observation that NSP4causes diarrhea in young rats. No group A rotavirus has been shown toinfect rats, suggesting that these animals lack the receptor for virusinfection. However, the induction of diarrhea with NSP4 indicates thereceptor for this protein is present and can be stimulated by NSP4resulting in an increase in intracellular calcium levels and disease.

These data collectively demonstrate that NSP4 is an enterotoxin: NSP4and NSP4 114-135 and 120-147 peptides induce diarrhea in two rodentmodels; diarrhea induction is specific, age- and dose-dependent; andelectrophysiologic analyses in Ussing chambers reveal that NSP4stimulates Cl⁻ secretion by a Ca²⁺-dependent pathway in young mouseintestinal mucosa. NSP4 interacts with an age-dependent intestinalreceptor, triggers a signal transduction pathway, and increases[Ca²⁺]_(i) resulting in Cl⁻ secretion or diarrhea.

EXAMPLE 22

Live Rotavirus and NSP4 Cause Diarrhea in CFTR Knock-out Mice.

Cystic Fibrosis is caused by a defect in the gene that codes for thecAMP-activated chloride channel called CFTR. As a result of the defect,the CFTR channel is defective and chloride secretion—and hence watersecretion—is greatly diminished. Without sufficient secretion of water,membranes accumulate excessive amounts of mucous and eventually becomeobstructed. We tested our theory that NSP4 stimulation of chloridesecretion through the alternate calcium-dependant chloride channel mightcompensate for the deficient secretion in Cystic Fibrosis patients. Weadministered peptide or virus to 5-7 day old CFTR knock-out mice—micehomozygous for a mutation that disables the CFTR coding region—and gotdiarrhea in 100% of the cases for virus and cross-linked peptide or in80% of the animals given 100 nmoles of non-crosslinked NSP4 114-135peptide. This demonstrates that NSP4 stimulation of chloride secretionthrough the Ca²⁺-dependent channel can compensate for the lack ofsecretion through the defective cAMP-dependent CFTR channel.

EXAMPLE 23

HIV gp120 Causes Diarrhea in Mice.

Human immunodeficiency virus (HIV) is associated with wasting or Slimdisease. To determine whether the HIV glycoprotein 120 (gp120) is anenterotoxin, 6-7 day old Balb/C mouse pups were inoculated with purifiedgp120. Diarrhea was observed in 100% of the animals. Other proteins ofHIV or other retrovirus or other proteins of other viruses may be foundto have similar functional activity—i.e., to directly induce diarrhea.

EXAMPLE 24

Identification of Small Molecule Inhibitors of NSP4/ReceptorInteraction.

The above data demonstrate that effective treatment of rotavirus-induceddiarrhea can be accomplished through inhibition of NSP4's interactionwith its receptor. Identification of small molecule inhibitors of NSP4is well within the ability of the ordinary practitioner according toknown techniques. Small molecule inhibitors are known in the art torefer to any ligand which can bind to a target molecule with sufficientaffinity to inhibit the target molecule's activity. Libraries of smallmolecules, such as random peptide libraries, random oligonucleotidelibraries, and pharmaceutical drug libraries, are available eitheraccording to known techniques or commercially, and may be quickly andeasily screened against a purified target molecule for small moleculesthat bind with high affinity to a target molecule. Examples include the“FliTrx Peptide Library,” (Invitrogen) and the SELEX technology.

EXAMPLE 25

Construction of Attenuated Rotavirus Strains by Incorporation of aSelected NSP4 Amino Acid Sequence.

The sequence of gene 10, the gene encoding NSP4, was determined for apair of virulent and tissue culture attenuated porcine rotavirusstrains. Double stranded RNAs were extracted from an intestinalhomogenate from a piglet infected with a virulent strain of porcinerotavirus (OSU-v) and from a piglet infected with a tissue cultureattenuated OSU virus (OSU-a). Gene 10 from the dsRNAs was amplified byRT/PCR using primers from the SA11 gene 10 sequence. cDNAs from thesetwo strains were cloned and sequenced. Comparisons of the gene 10sequences of these two strains and other rotavirus strains havesuggested that the amino acid sequence between amino acids 131 to 140are important in pathogenesis. The amino acid sequence of the NSP4protein from the attenuated strain (OSU-a) (SEQ ID NO. 1) was comparedto that of the virulent strain (OSU-v) (SEQ ID NO. 8) and the resultsare presented in FIG. 7. The positions at which the two sequences differare shown in bold. Mice infected with virulent virus develop diarrheawhile those infected with attenuated virus do not.

Gene 10 encoding NSP4 protein from each of these two strains has beencloned and expressed in a baculovirus expression system and purified.The purified NSP4 proteins were tested for their ability to inducediarrhea in mouse pups. The NSP4 protein from the virulent strain causesincreased intracellular calcium concentration and induced diarrhea whilethat of the attenuated strain did not. These results indicate thatavirulence is associated with mutations in gene 10 and indicate thatcertain amino acid positions of the NSP4 protein are critical fordiarrhea induction. The identification of critical residues makes it aroutine matter for one skilled in the art to determine whether a givenrotavirus is likely to cause diarrhea by comparing the amino acidsequence of the NSP4 protein to known sequences. In addition, theidentification of NSP4 sequences that correlate to an attenuatedphenotype makes it a routine matter to construct attenuated reassortmentviruses that include such an NSP4 sequence, using techniques that arewell known to those skilled in the art. This permits the construction ofrotaviruses for use as vaccines that retain the antigenicity of thevirulent strain yet display an attenuated phenotype as a result of theincorporation into the genome of the virus a nucleic acid coding for anNSP4 protein having a selected sequence.

EXAMPLE 26

Preparation and Use of an NSP4 Toxoid.

Vaccines comprising NSP4 in the form of a toxoid may be prepared frompurified NSP4 protein. The purified protein can be chemically treated,using known techniques, to inactivate the biological activity of theNSP4 protein while retaining the immunogenicity. For example, thepurified protein may be treated with a 10% solution of formaldehyde atabout 37 degrees for about an hour. One skilled in the art willrecognize that other equivalent protocols to produce a toxoid may beemployed without deviating from the spirit of the invention. Afterchemical treatment the toxoid will typically be washed with buffer, forexample phosphate buffered saline or the like, and formulated into avaccine. The toxoid may be in solid form such as adsorbed to alum or thelike. Alternatively, the toxoid may be in solution in anypharmaceutically acceptable liquid. The toxoid may be administered as avaccine in the absence of adjuvant. A vaccine formulated with the toxoidmay include adjuvants including but not limited to alum, Freund'scomplete and incomplete adjuvants, Ribi's adjuvant, bacterial andmycobacterial cell wall components and derivatives thereof, liposomesand any other adjuvant formulation known in the art. Vaccines thusformulated may be administered using parenteral or mucosal routes suchas by intraperitoneal, intranasal, intragastric, subcutaneous,intramuscular, or rectal application.

EXAMPLE 27

Characterization of the Receptor for NSP4.

The human intestinal cell line HT29 was assayed for sensitivity to NSP4.In response to purified NSP4, these cells showed an increase inintracellular calcium levels. When these cells are pre-treated withtrypsin, the response is ablated. The binding of radiolabelled NSP4protein to responsive cells is dose-dependent and saturable as would beexpected for a receptor dependent phenomenon. Taken together, these tworesults demonstrate that NSP4 binds to a protein receptor. Recent testswith respiratory epithelial cells have demonstrated that these cells donot respond to NSP4 and do not bind radiolabelled NSP4 protein. It iswell within the ability of one of ordinary skill in the art to identifythe receptor by expression cloning in these nonresponsive cells that donot bind NSP4. The mRNA from a responsive cell can be isolated usingstandard techniques and reverse transcribed into cDNA. This cDNA canthen be inserted into a vector and then used to transform thenonresponsive cell line. Alternatively, the genomic DNA from theresponsive cells may be inserted into a vector and used to transform thenonresponsive cell line. The transformed cells will be screened for theexpression of the receptor using routine techniques, for example, byscreening for cells capable of binding radiolabelled NSP4. Cells thatexpress the receptor will be isolated.

The presence compounds that inhibit the G-protein/phospholipase C signaltransduction pathway also abrogate the intracellular calcium increaseseen in response to NSP4, suggesting the involvement of this pathway inthe observed response. One skilled in the art can envision the use ofinhibitors specific for the G-protein/phospholipase C pathway astherapeutic agents for the treatment of NSP4 induced diarrhea. The useof such compounds is within the spirit of the present invention.

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The invention disclosed herein is not considered to be limited by anystatements made herein.

TABLE 1 Peptide Sequence¹ AS² Pt³ Mr⁴ NSP4 114-135DKLTTREIEQVELLKRIYDKLT 35 1.12 (YDKL) 2705 NSP4 2-22EKLTDLNYTLSVITLMNNTLH 13.9 1.12 (TDLN) 2434 NSP4 90-123TKDEIEKQMDRVVKEMRRQLEMIDKLTTREIEO 70.6 1.11 (TKDE) 4092 NSP4 m131KDKLTTREIEQVELLKRI(K)DKLT 31.4 1.08 (KDKL) 2669 NV 464-483KTGRNLGEFKAYPDGFLTCV 41.4 1.58 (YPDG) 2204 ¹NSP4 sequence from rotavirusSA11 (Both et al., 1983). Norwalk virus (NV) sequence from Jiang et al.,1990. Underlined sequence is the region of the NSP4 2-22 peptide whichoverlaps with the NSP4 114-135 peptide. This substitution decreases theturn potential from 1.12 to 1.08. The mutated tyrosine to lysine residueis shown in bold and in parentheses. ²AS = amphipathic score. A blocklength of 11 was used with an AS of 4 considered significant (Margolitet al., 1987). ³Pt = turn potentials greater than 1.0 within theselected peptide based on the algorithm of Chou and Fasman (1974, 1978).⁴Mr = Theoretical mass.

TABLE 2 Intraileal administration of NSP4 and NSP4 114-135. AgeConcentration Species (days) Inoculum (nmol) Diarrhea Sprague-Dawley rat6-7 X-linked NSP 4 114-135 120-240  9/10 Balb/C mice 8-9 X-linked NSP 4114-135 10 6/6 Balb/C mice 11-12 X-linked NSP 4 114-135 10 2/6 Balb/Cmice 15-17 X-linked NSP 4 114-135 10 0/6 CD1 mice  7 X-linked NSP 4114-135 50 3/5 CD1 mice 11-12 X-linked NSP 4 114-135 50 2/8 CD1 mice17-18 X-linked NSP 4 114-135 50 0/6 CD1 mice 25 X-linked NSP 4 114-13550 0/5 CD1 mice 25 X-linked NSP 4 114-135 100-200 0/8 CD1 mice 8-9 NSP4protein 0.5 5/5

TABLE 3 Specificity of the Diarrheal Response: Diarrhea Induction in CD1Mice following IP Administration of 50-100 nmol Peptide Peptide %Diarrhea # responders/total # tested NSP4 114-135 67  8/12antibody^(a) + NSP4 114-135 10  1/10 cross-linked NSP4 114-135 75 15/20mNSP4 131K^(b)  0  0/11 cross-linked mNSP4 131K 20  2/10 NSP4 2-22  0 0/11 cross-linked NSP4 2-22  0  0/17 NSP4 90-123 20  2/10 cross-linkedNSP4 90-123 50 4/8 NV 463-486  0  0/10 cross-linked NV 463-486  0 0/9^(a)rabbit hyperimmune anti-NSP4 114-135 serum administered just priorto administration of the peptide ^(b)single amino acid substitution atresidue 131

TABLE 4 Electrophysiological Analyses of CD1 Mice Ileal Mucosa 19-22 DayOld Mice 35 Day Old Mice Agonist Treatment^(a) ▴I_(SC) (μA/cm²)^(b)▴I_(SC) (μA/cm²)^(b) Forskolin (FSK), 5 μM  44 ± 0.7 (n = 8) 41 ± 7  (n= 5) Carbachol (Cch), 5 μM 9 ± 2 (n = 8) 14 ± 4  (n = 5) NSP4 114-135peptide, 5 μM^(c)   3 ± 0.2 (n = 4) 0.4 ± 0.4 (n = 4)^(d) FSK (5 μM) =Cch (5 μM) 63 ± 10 (n = 5) 64 ± 9  (n = 6) FSK (5 μM) + NSP4 114-135peptide (5 μM) 64 ± 5  (n = 7) 43 ± 9  (n = 5) ^(a)The mean restingconductance for the ileal mucosal sheets prior to agonist treatment was10.4 ± 4.8 msemens (ms)/cm² (n = 32) for the 19-22 day old mice and 12.3± 3.8 ms/cm² (n = 25) for the 35 day old mice. ^(b)The ▴I_(SC) wascalculated by subtracting the stimulated I_(SC) measurement from theI_(SC) measured immediately before the addition of agonist. All agoniststimulated values were significantly different (p < 0.001, unpairedt-test). ^(c)NSP4 114-135 peptide is active when added to either surfaceof the mucosa. ^(d)For n = 3, there was no response with peptide; for n= 1, the response was 2 μA/cm²

TABLE 5 Immunizaton with NSP4 114-135 peptide induces protectiveimmunity from infectious rotavirus challenge Diarrhea Observed inPassively Immunized Pups^(a) % pups with diarrhea^(b) Mean Peptide total2 days 3 days Diarrhea Score NSP4 2-22 100% 63% 25% 3.5+ (16/16) (10/16)(4/16) NSP 4 114-135  42% 17%  0% 2.0+  (5-12)  (2/12) (0/12) *P =<0.001 P = <0.025 NS *Fischer's Exact Test ^(a)Pups born to damsimmunized with NSP4 114-135 or control peptide were challenged with ahigh dose of infectious SA11 rotavirus and diarrheal disease wasmonitored. ^(b)Significant protection against disease was seen in pupsborn to mothers immunized with NSP4 114-135 peptide.

TABLE 6 Protection from Rotavirus Severe Diarrhea (≧3+) afterAdministration of NSP4-specific Antibody Total # with Onset of Illnessat Illness at Treatment Diarrhea Diarrhea (hpi) 71-77 hpi 90-110 hpiNone Exp. 1 7/7 (100%) 27-28 (5/7 4/7 (57%) 2/7 (29%) Exp. 2 10/10(100%) 22-23 6/10 4/10 (40%) 3/10 (30%) Rab pre immune Exp. 1 8/8 (100%)27-28 5/8 4/8 (50%) 0/8 (0%) NRS Exp. 2 13/13 (100%) 22-24 3/14 2/13(15%) 0/13 (0%) Rabbit anti-NSP4 Exp. 1 5/10* (50%) 27-28 3/10 1/8 (12%)0/8 (0%) Exp. 2 2/13* (15%) 27-28 1/13 0/10 (0%) 0/10 (0%) Rabbitanti-NSP4 Exp. 1 ND ND ND ND anti-NSP4 Exp. 2 2/9* (22%) 22-23 1/9 0/9(0%) 0/9 (0%) 114-135 peptide *Statistically significant compared to thepre immune or no treatment groups; Fischer's exact (2-tailed). ND = notdone.

8 1 22 PRT Artificial Sequence Description of Artificial SequenceSynthetic NSP4-specific control peptide 1 Asp Lys Leu Thr Thr Arg GluIle Glu Gln Val Glu Leu Leu Lys Arg 1 5 10 15 Ile Tyr Asp Lys Leu Thr 202 21 PRT Artificial Sequence Description of Artificial SequenceSynthetic NSP4-specific control peptide 2 Glu Lys Leu Thr Asp Leu AsnTyr Thr Leu Ser Val Ile Thr Leu Met 1 5 10 15 Asn Asn Thr Leu His 20 333 PRT Artificial Sequence Description of Artificial Sequence SyntheticNSP4-specific control peptide 3 Thr Lys Asp Glu Ile Glu Lys Gln Met AspArg Val Val Lys Glu Met 1 5 10 15 Arg Arg Gln Leu Glu Met Ile Asp LysLeu Thr Thr Arg Glu Ile Glu 20 25 30 Gln 4 22 PRT Artificial SequenceDescription of Artificial Sequence Synthetic NSP4-specific controlpeptide 4 Asp Lys Leu Thr Thr Arg Glu Ile Glu Gln Val Glu Leu Leu LysArg 1 5 10 15 Ile Lys Asp Lys Leu Thr 20 5 20 PRT Artificial SequenceDescription of Artificial Sequence Synthetic NSP4-specific controlpeptide 5 Asp Thr Gly Arg Asn Leu Gly Glu Phe Lys Ala Tyr Pro Asp GlyPhe 1 5 10 15 Leu Thr Cys Val 20 6 28 PRT Artificial SequenceDescription of Artificial Sequence Synthetic NSP4-specific controlpeptide 6 Glu Ile Glu Gln Val Glu Leu Leu Lys Arg Ile Tyr Asp Lys LeuThr 1 5 10 15 Val Gln Thr Thr Gly Glu Ile Asp Met Thr Lys Glu 20 25 7175 PRT Porcine rotavirus 7 Met Asp Lys Leu Ala Asp Leu Asn Tyr Thr LeuSer Val Ile Thr Leu 1 5 10 15 Met Asn Asp Thr Leu His Ser Ile Ile GlnAsp Pro Gly Met Ala Tyr 20 25 30 Phe Pro Tyr Ile Ala Ser Val Leu Thr ValLeu Phe Thr Leu His Lys 35 40 45 Ala Ser Ile Pro Thr Met Lys Ile Ala LeuLys Thr Ser Lys Cys Ser 50 55 60 Tyr Lys Val Ile Lys Tyr Cys Met Val ThrIle Ile Asn Thr Leu Leu 65 70 75 80 Lys Leu Ala Gly Tyr Lys Glu Gln ValThr Thr Lys Asp Glu Ile Glu 85 90 95 Gln Gln Val Asp Arg Ile Ile Lys GluMet Arg Arg Gln Leu Glu Met 100 105 110 Ile Asp Lys Leu Thr Thr Arg GluIle Glu Gln Val Glu Leu Leu Lys 115 120 125 Arg Ile His Asp Lys Leu AlaAla Arg Ser Val Asp Ala Ile Asp Met 130 135 140 Ser Lys Glu Phe Asn GlnLys Asn Ile Arg Thr Leu Asp Glu Trp Glu 145 150 155 160 Ser Gly Lys AsnPro Tyr Glu Pro Ser Glu Val Thr Ala Ser Met 165 170 175 8 175 PRTPorcine rotavirus 8 Met Asp Lys Leu Ala Asp Leu Asn Tyr Thr Leu Ser ValIle Thr Leu 1 5 10 15 Met Asn Asp Thr Leu His Ser Ile Ile Gln Asp ProGly Met Ala Tyr 20 25 30 Phe Pro Tyr Ile Ala Ser Val Leu Thr Val Leu PheThr Leu His Lys 35 40 45 Ala Ser Ile Pro Thr Met Lys Ile Ala Leu Arg ThrSer Lys Cys Ser 50 55 60 Tyr Lys Val Ile Lys Tyr Cys Ile Val Thr Ile IleAsn Thr Leu Leu 65 70 75 80 Lys Leu Ala Gly Tyr Lys Glu Gln Val Thr ThrLys Asp Glu Ile Glu 85 90 95 Gln Gln Met Asp Arg Ile Val Lys Glu Met ArgArg Gln Leu Glu Met 100 105 110 Ile Asp Lys Leu Thr Thr Arg Glu Ile GluGln Val Glu Leu Leu Lys 115 120 125 Arg Ile His Asp Lys Leu Val Val ArgPro Val Asp Ala Ile Asp Met 130 135 140 Ser Lys Glu Phe Asn Gln Lys AsnIle Arg Thr Leu Asp Glu Trp Glu 145 150 155 160 Ser Gly Lys Asn Pro TyrGlu Pro Ser Glu Val Thr Ala Ser Met 165 170 175

We claim:
 1. A method of immunization against rotavirus infection ordisease comprising administering to a subject a peptide NSP4 114-135 ora toxoid thereof.
 2. A method for passive immunization against rotavirusinfection or disease comprising administering to an expectant mother apeptide NSP4 120-147 or toxoid thereof.
 3. A method of immunizationagainst rotavirus infection or disease comprising administering to asubject a peptide NSP4 120-147 or a toxoid thereof.
 4. A method forpassive immunization against rotavirus infection or disease comprisingadministering to an expectant mother a peptide NSP4 114-135 or a toxoidthereof.
 5. The method of claim 1 or 3, wherein said peptide or toxoidthereof is produced by a synthetic method.
 6. The method of claim 1 or 3wherein said peptide or toxoid thereof is produced by an expressionvector.